This invention relates to the identification and investigation of receptors having affinity for selected vectors.
It is well known that particular biological receptors such as cell receptors may bind certain vectors, also known as ligands or binding partners, often with a high degree of specificity. Considerable attention is currently being given to the preparation of synthetic peptides and other vectors which target disease-specific markers in vivo, for example as potential components of targeted imaging agents and/or therapeutic agents. Once a vector is found to have appropriate targeting properties, for example affinity for endothelial or other target tissue, it is desirable to identify the receptor to which it is binding.
The present invention is based on the finding that receptor identification may be achieved relatively quickly, simply and cheaply using a retroviral cDNA library containing activated genes from the target tissue to transfect an appropriate cell line and separating cells which express the receptor of interest using encapsulated gas microbubbles which are coupled to the vector under investigation. The cDNA encoding this receptor may then be amplified, e.g. using the polymerase chain reaction, and identified, for example by sequencing and comparison of the sequence with sequences in a database e.g. of known genes.
The fact that a wide range of vectors may be bound to microbubbles is a very important advantage of the invention.
Mammalian cells transfected with retroviral cDNA libraries have previously been employed in a variety of identification techniques primarily based on phenotypical changes of cells caused by the transfected gene. For example, Nicholson et al. in J. Leukoc. Biol. 63(6), pp. 665-668 (1998) describe the identification of a new family of negative regulators of signal transduction by selecting cells that were no longer able to differentiate in response to interleukin-6. Cell surface molecules have been identified through the transfection of a cell line with a cDNA retroviral library; thus Tailor et al. in J. Virol. 73(5), pp. 4470-4474 (1999) describe the identification of a cell surface receptor for type D simian viruses after retroviral transfection using human T-lymphocyte cDNA.
Zannettino et al. in J. Immunol. 156(2), pp. 611-620 (1996) describe a method for rapidly isolating genes encoding cell surface molecules from a human bone marrow stromal cell cDNA library constructed in a retroviral vector. A selection strategy using antibody (e.g. monoclonal antibody)-coated magnetic beads is employed to isolate cells expressing particular cell surface antigens; cDNA encoding the selected cell surface molecules is recovered using the polymerase chain reaction and is subjected to sequencing.
Separation of cells using magnetic beads, e.g. antibody-coated superparamagnetic polymer particles such as Dynabeads(copyright), suffers from a number of disadvantages. Firstly, when a magnetic field is applied to a sample, magnetic beads and target cell/bead complexes will rapidly be drawn through the sample towards the magnet. Since beads such as polymer particles are hard and may typically be of similar size to cells, this rapid movement can cause significant damage to target cells in the sample.
Moreover, detachment of separated cells from the beads can be a difficult and time-consuming process. A representative technique where the beads are coated with a monoclonal antibody having specificity for the target cells involves use of a polyclonal antibody that reacts with Fab-fragments of monoclonal antibodies to effect direct dissociation of the antigen-antibody binding between the beads and the target cells. This technique is only suitable for use with certain types of polymer particle and certain monoclonal antibodies. Alternative detachment methods include overnight incubation at 37xc2x0 C., enzymatic cleavage and the introduction of reagents which compete for the same target as the beads.
Due to the complicated nature of these detachment techniques, magnetic beads are predominantly employed in xe2x80x9cnegativexe2x80x9d component separations, i.e. processes in which unwanted components are bound to the magnetic beads and isolated, leaving the desired components in the sample. Their use in xe2x80x9cpositivexe2x80x9d selections where a specific component is targeted and isolated, as in the procedure of Zannettino et al., is comparatively limited.
Gentle positive selection is important when the cells expressing the particular receptor are going to be withdrawn for further growth. Flotation separations as used in accordance with the present invention, in which cells expressing the receptor of interest become bound to vectors coupled to surfaces of encapsulated gas microbubbles, have the advantage that the efficiency of such separations is enhanced by the substantial density difference between gas microbubbles and liquid sample media, so that the process is capable of high sensitivity. Flotation separations inherently proceed more gently than magnetic separations and the gas microbubbles may advantageously be prepared using flexible encapsulating materials, so that the possibility of causing damage to sensitive cells during separation may thus be minimised. The use of encapsulated gas microbubbles also permits ready removal of the microbubbles from the cells after separation, simply by bursting the microbubbles. This is of particular advantage in a positive selection process, and encapsulated microbubbles coupled to a wide range of vectors may be used in the process of the invention; it is therefore far more widely applicable than the procedure of Zannettino et al., which is limited to the use of antibodies as vectors.
Thus according to one aspect of the invention there is provided a method for the identification of a receptor in target tissue for which a selected vector has affinity, said method comprising:
i) creating retroviral particles containing a library of mRNA from the target tissue;
ii) transfecting a non-adherent cell line which does not bind with the selected vector by infecting the cells with said retroviral particles;
iii) adding to the transfected cell line a suspension of encapsulated microbubbles to which the selected vector is coupled and allowing the microbubbles and cells coupled thereto to float to the surface of the suspension;
iv) isolating the microbubble-bound cells at the surface;
v) lysing the isolated cells, amplifying the receptor-encoding cDNA therefrom and sequencing said cDNA; and optionally
vi) comparing the thus-obtained cDNA sequence data with gene bank sequence data.
In an alternative embodiment there is provided a method for the investigation of a receptor as defined above in which the isolated cells from step (iv) are cultured, rather than lysed, to allow further study of the nature of the receptor to take place, e.g. studies on affinity between the isolated cells and potential vectors.